Raffinose saccharides are a group of D-galactose-containing oligosaccharide derivatives of sucrose that are widely distributed in plants. Raffinose saccharides are characterized by the general formula: [O-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n -.alpha.-glucopyranosyl-(1.fwdarw.2)-.beta.-D-fructofuranoside where n=0 through n=4 are known respectively as sucrose, raffinose, stachyose, verbascose, and ajugose.
Extensive botanical surveys of the occurrence of raffinose saccharides have been reported in the scientific literature [see Dey (1985) in Biochemistry of Storage Carbohydrates in Green Plants, P. M. Dey and R. A. Dixon, Eds. Academic Press, London, pp. 53-129]. Raffinose saccharides are thought to be second only to sucrose among the nonstructural carbohydrates with respect to abundance in the plant kingdom. In fact, raffinose saccharides may be ubiquitous, at least among higher plants. Raffinose saccharides accumulate in significant quantities in the edible portion of many economically-significant crop species. Examples include soybean (Glycine max L. Merrill), sugar beet (Beta vulgaris), cotton (Gossypium hirsutum L.), canola (Brassica sp.) and all of the major edible leguminous crops including beans (Phaseolus sp.), chick pea (Cicer arietinum), cowpea (Vigna unguiculata), mung bean (Vigna radiata), peas (Pisum sativum), lentil (Lens culinaris) and lupine (Lupinus sp.).
Although abundant in many species, raffinose saccharides are an obstacle to the efficient utilization of some economically-important crop species. Raffinose saccharides are not digested directly by animals, primarily because .alpha.-galactosidase is not present in the intestinal mucosa [Gitzelmann et al. (1965) Pediatrics 36:231-236; Rutloff et al. (1967) Nahrung 11:39-46]. However, microflora in the lower gut are readily able to ferment the raffinose saccharides resulting in an acidification of the gut and production of carbon dioxide, methane and hydrogen [Murphy et al. (1972) J. Agr. Food. Chem. 20: 813-817; Cristofaro et al. (1974) in Sugars in Nutrition, H. L. Sipple and K. W. McNutt, Eds. Academic Press, New York, Chap. 20, 313-335; Reddy et al. (1980) J. Food Science 45:1161-1164]. The resulting flatulence can severely limit the use of leguminous plants in animal, particularly human, diets. It is unfortunate that the presence of raffinose saccharides restricts the use of legumes in human diets because many of these species are otherwise excellent sources of protein and soluble fiber. Varieties of edible beans free of raffinose saccharides would be more valuable for human diets and would more fully use the desirable nutritional qualities of edible leguminous plants.
Soybean meal is the principal source of protein in animal feed, especially feed for monogastric animals such as poultry and swine. Approximately 28 million metric tons of soybean meal were produced in the U.S. in 1988 [Oil Crops Situation and Outlook Report (April 1989) U.S. Dept. of Agriculture, Economic Research Service]. Soybean meal is produced by treating soybeans with hexane to remove the oil and then toasting the extracted material to remove the residual solvent. Although the soybean is an excellent source of vegetable protein, there are inefficiencies associated with its use that appear to be due to the presence of raffinose saccharides. Compared to maize, the other primary ingredient in animal diets, gross energy utilization for soybean meal is low [see Potter et al. (1984) in Proceedings World Soybean Conference III, 218-224]. For example, although soybean meal contains approximately 6% more gross energy than ground yellow corn, it has about 40 to 50% less metabolizable energy when fed to chickens. This inefficiency of gross energy utilization does not appear to be due to problems in digestion of the protein fraction of the meal, but rather due to the poor digestion of the carbohydrate portion of the meal. It has been reported that removal of raffinose saccharides from soybean meal by ethanol extraction results in a large increase in the metabolizable energy for broiler chickens [Coon et al. (1988) Proceedings Soybean Utilization Alternatives, University of Minnesota, 203-211]. Removal of the raffinose saccharides was associated with increased utilization of the cellulosic and hemicellulosic fractions of the soybean meal. Soybean varieties free of raffinose saccharides could be used to produce meals that would have added value for individuals who either produce soybean meal for animal feed or use soybean meal as a major component in the diets for their animals.
In addition to its use in animal diets, soybeans are used to produce enriched sources of vegetable protein for human use. Examples of soybeans in human foods include soy protein concentrate, textured soy protein and infant formula. Facilities and methods to produce protein isolates from soybeans are available across the U.S. One of the unsolved challenges faced by producers of soy protein isolates is selectively purifying the protein away from the raffinose saccharides. Considerable added costs result from removing the large amounts of raffinose saccharides that are present in soybeans. Again, soybean varieties free of raffinose saccharides would reduce the cost of producing soy protein products as well as improve the nutritional quality of the end product.
Other agronomically-important crops such as cotton and canola are also used as secondary sources of protein for animal diets. Meals produced from seeds of these species also contain raffinose saccharides. The effect of raffinose saccharides on the nutritional quality of cottonseed and canola meal has received little or no attention, but it is possible that raffinose saccharides are as great a barrier to the use of these meals as they appear to be with soybean. Both cotton and canola meal have less value than soybean meal for metabolizable energy to animals [Feedstuffs (1990) Reference Issue 62:24-31]. Varieties of cotton or canola free of raffinose saccharides may have added value for use in animal feed.
An additional problem associated with the presence of raffinose in plants occurs in the production of sucrose from sugar beets. The small amount of raffinose, ca. 0.05%, compared to sucrose, ca. 16%, in expressed beet juice is sufficient to decrease the efficiency of crystallization of sucrose. As a result, sugar manufacturers have resorted to the use of immobilized .alpha.-galactosidase to reduce the content of molasses during the refining of sugar beet juice [Linden (1982) Enzyme Microb. Technol. 4:130-136]. Sugar beet varieties free of raffinose would not need this additional processing and therefore would have added value to sugar beet processors.
Although nutritional and economic problems are associated with the presence of raffinose saccharides in many crops, certain benefits are also ascribed to this family of oligosaccharides. Seed viability has been correlated positively with the presence of raffinose [Ovacharov et al. (1974) Fiziol. Rast. 21:969-974; Caffrey et al. (1988) Plant Physiol. 86:754-758; Schleppi et al. (1989) Iowa Seed Science 11:9-12]. It is thought that raffinose helps maintain the integrity of the membranes of seeds as they undergo the desiccation process during maturation. Raffinose also may play an important role in the cryoprotection of plants. The accumulation of raffinose in plants exposed to cold temperature has been indicated in a number of species [Parker (1959) Bot. Gaz. 121:46-50; Alden et al. (1971) Bot. Rev. 37:37-142, see Kandler et al. (1982) in Encyclopedia of Plant Physiology, New Series, Vol. 13A:348-383; Mitcham-Butler et al. (1987) J. Amer. Soc. Hort. Sci. 112:672-676; Castillo et al. (1990) J. Agric. Food Chem. 38:351-355]. This accumulation of raffinose may be responsible for protection of chloroplasts and has been shown to be highly correlated with the postharvest retention of needles in horticulturally-important coniferous species such as Fraser fir [Abies fraseri (Pursh) Poir.] and white pine (Pinus strobus L.). Retention of needles in these species affects their quality for use as ornamental plants, such as Christmas trees. Producing plants with increased amounts of raffinose saccharides may increase cold hardiness resulting in increased post-harvest quality, a greater ability to withstand cold temperatures or provide producers greater flexibility when harvesting trees.
In spite of the problems associated with the presence of raffinose saccharides in soybean products for human use, for certain food applications increasing the amount of fermentable carbohydrates present in the soybean offers some advantages. [Economic Implications of Modified Soybean Traits (1990) Special Report ISSN: 0361-199X Iowa State Report]. Examples include oriental foods such as tofu, tempeh, natto, and soy sauce. In these applications, soybean varieties with increased amounts of readily-fermentable sugars such as sucrose and the raffinose saccharides would have added value for the producers of these products.
The biosynthesis of raffinose saccharides has been fairly well characterized [see Dey (1985) in Biochemistry of Storage Carbohydrates in Green Plants, P. M. Dey and R. A. Dixon, Eds. Academic Press, London, pp. 53-129]. The committed reaction of raffinose saccharide biosynthesis involves the synthesis of galactinol from UDP-galactose and myo-inositol. The enzyme that catalyzes this reaction is galactinol synthase. Synthesis of raffinose and higher homologues in the raffinose saccharide family from sucrose is thought to be catalyzed by distinct galactosyltransferases (for example, raffinose synthase and stachyose synthase). Studies with many species suggest that galactinol synthase is the key enzyme controlling the flux of reduced carbon into the biosynthesis of raffinose saccharides [Handley et al. (1983) J. Amer. Soc. Hort. Sci. 108:600-605; Saravitz, et al. (1987) Plant Physiol. 83:185-189]. Altering the activity of galactinol synthase, either as a result of overexpression or through antisense inhibition, would change the amount of raffinose saccharides produced in a given tissue.
In order to alter the activity of galactinol synthase using molecular biological approaches it is essential to isolate the gene(s) or cDNA(s) encoding the enzyme. There are no published reports for the purification of homogeneous galactinol synthase which would allow one to prepare DNA probes based on amino acid sequence information. Applicants describe here the purification of galactinol synthase from zucchini (Cucurbita pepo) and the subsequent cloning and use of galactinol synthase-encoding nucleotide sequences from zucchini, soybean, and canola.